Methods and molecules useful for identifying molecules that bind LFA-1 and for determining receptor occupancy
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example 1
LFA-1 was purified using the TS2 / 4 mAb (American Type Culture Collection; Manassas, Va.) from a 20 g pellet of human JY or SKW3 cells, utilizing a protocol previously described (Dustin, M. J. et al., 1992, J. Immunol. 148:2654-2660). The LFA-1 was purified from SKW3 lysates by immunoaffinity chromatography on TS2 / 4 LFA-1 mAb Sepharose and eluted at pH 11.5 in the presence of 2 mM MgCl2 and 1% octylglucoside. After collection and neutralization of fractions from the TS2 / 4 column, samples were pooled and precleared with Protein G agarose.
Micellar LFA-1 (50 μl) was immobilized onto a microtiter plate by adsorption at approximately 3 μg / ml in assay buffer (AB: dPBS+2 mM Mg++) for 1 hr at room temperature. Non-specific sites were then blocked for 30 min with 2% BSA-AB. Various purified mAbs (50 μl at 10 μg / ml) (CLB-LFA-1, Research Diagnostics Inc., Flanders, N.J.; R7.1 and R3.1, generated at Boehringer Ingelheim Pharmaceuticals, Inc. (BIPI), Ridgefield, Conn.; MHM 24, Biomedia, Foster Ci...
example 2
SKW-3 Cells
Using flow cytometry, the binding of R3.1 to SKW-3 cells, a T-cell lymphoma that expresses high amounts of LFA-1, was assessed. After washing and counting, 100 μl of SKW-3 cells (1×107 cells / ml) were incubated with 13.3 nM mAb38 or R3.1 or R15.7 (generated at BIPI, Ridgefield, Conn.) and 10 μM Compound 1 or DMSO (0.09%) (negative control) for 30 minutes at 37° C. After centrifugation and 2 washes in HBSS (GIBCO, Grand Island, N.Y.), cells were incubated with a goat anti-mouse IgG-PE conjugate (Biosource International, Camarillo, Calif.) (diluted 1:50 in dPBS) for 20 minutes at 4° C. After 2 washes in dPBS, cells were fixed with 500 μl 1% paraformaldehyde and analyzed on a FACScan (Becton Dickinson, San Jose, Calif.).
Results
FIG. 2 shows that Compound 1 but not DMSO inhibited the binding of R3.1 to the cell-bound LFA-1. Similar to the molecular assay described in Example 1, Compound 1 did not inhibit the binding of mAb38, another I domain binding mAb) or the anti-CD 18 mAb,...
example 3
Human Whole Blood Cells
Using flow cytometry, the binding of R3.1 to human whole blood cells was assessed. Peripheral blood was obtained from normal healthy donors by venipuncture. R3.1 (3.3 nM) or R7.1 (3.3 nM) and various dilutions of Compound 1 or DMSO (0.2%) were incubated with 100 μl of human whole blood for 20 minutes at 4° C. After centrifugation and 1 wash in dPBS, the cells were then incubated with a goat anti-mouse IgG-PE conjugate (Biosource International, Camarillo, Calif.) diluted 1:50 in dPBS. After incubation in secondary reagent for 15 minutes at 4° C., cells were lysed and fixed in 100 μl of lysis buffer (Immunotech, Westbrook, Me.) for 10 min at room temperature and then 1 ml of water was added to each sample before analysis on a FACScan (Becton Dickinson, San Jose, Calif.).
Results
FIG. 3 shows that Compound 1, but not DMSO, inhibited the binding of R3.1 to leukocytes in a dose dependent manner. The same concentrations of compound did not inhibit the binding of the a...
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